Genetic organization of the two arsenic resistance clusters in strain O. tritici SCII24. Gene orientations are shown by arrows. Within the predicted structure of the promoters, the -35, -10 regions and ribosome binding sites (RBS) are boldfaced and ATG codons are in boxes.

Figure 2

Alignment of ArsR (A) and ArsC (B) proteins. The three ArsR sequences from O. tritici were aligned with ArsR of E. coli pR773 (P15905). Both ArsCs from O. tritici were aligned with ArsC homologues from E. coli pR773 (AAA21096) and Staphylococcus aureus pI258 (AAA25638). The multiple alignment was calculated with CLUSTAL W.

Expression of the O. tritici ars genes in E. coli AW3110 under control of ptrc promoter. The gel used was a SDS-12% polyacrylamide gel. Lane1, marker proteins, lane2, plasmid pTRC 99A without an insert; lanes 3, 4 and 5, construct parsRDAcbsB in absence of any oxyanion, in presence of 1 mM As(III) and 1 mM Sb(III), respectively; lanes 6, 7 and 8, construct parsDAcbsB without any oxyanion, in presence of 1 mM As(III) and 1 mM Sb(III), respectively.

However, this protein was not found when the same clone was grown in the absence of arsenite or antimonite. Furthermore, all different clones without arsR1 gene cultivated in the presence or absence of these chemicals were always able to produce this ArsA protein, indicating that ArsR worked as a repressor protein in E. coli. In contrast, all of the others ars products (ArsR1, ArsD, CBS domain, ArsB, ArsR2, ArsC1, ACR3, ArsC2, ArsH and ArsR3) were not found in the total proteins of E. coli.

Ability of the cloned

Different constructs of the ars genes were used to evaluate the role of these genes in arsenic resistance in E. coli AW3110. All experiments were done in the presence of 0.5 mM IPTG as inducer and the cultures were analyzed for their ability to grow on increasing concentrations of arsenite, arsenate and antimonite (Fig. 4). Cells carrying the constructs from ars1 operon parsRDAcbsB, parsDAcbsB, parsAcbsB and parscbsB showed equivalent levels of resistance to As(III) (Fig. 4A). On the other hand, resistance to arsenite was not found in cells carrying the emptyvector, or containing the constructs with each of the following genes: arsA (parsA), arsB (parsB), cbs (pcbs), arsA and cbs (parsAcbs).

Figure 4

Growth of E. coli AW3110 containing different constructs in the presence of arsenite (A and B), arsenate (C) and antimonite (D). Arsenite and antimonite resistance assays were performed in LB medium and arsenate growths were performed in low-phosphate medium. Each data point represents the results of three independent assays. The error bars indicate standard deviations. O.D. 600 nm, optical density at 600 nm.

The experiments related to arsenite resistance by ars2 operon, showed that this second operon also conferred As(III) resistance to E. coli cells, but at a lower level than the resistance conferred by operon ars1. Comparing the different constructs of ars2 operon (Fig. 4B), the clone with all genes (parsR2C1Acr3C2HR3) showed the highest As(III) resistance. Cells carrying the constructs parsR2C1Acr3C2H, parsAcr3C2HR3 and parsAcr3C2H showed equivalent levels of resistance to As(III). The constructs parsAcr3C2 and parsAcr3 conferred similar resistance, although at a lower level than the previous constructs and this was more evident for high As(III) concentrations. When the experiments were conducted in the presence of arsenate, we observed that only the cloned ars2genes increased the level of resistance of the host strain. Therefore, abilities of parsR2C1Acr3C2HR3 and subclones to confer resistance to arsenate were tested (Fig. 4C). All the combinations of the multiplears genes tested conferred similar levels of resistance to As(V). Constructs containing the individual genes did not confer resistance to the host strain except for cells that expressed the arsC gene, which showed a slight resistance to this oxyanion. These experiments are in agreement with the notion that arsCs encode for arsenate reductases, involved in As(V) detoxification. The ars1 operon was also responsible for antimony resistance, but the ars2 operon was not able to confer resistance to this oxyanion. The figure4D shows that clones, parsDAcbsB, parsAcbsB and parscbsB conferred Sb(III) resistance to E. coli cells, as well as the construct parsRDAcbsB although at an intermediary level. Clone carrying only arsB grew less than the wild type in the absence of metal and was not resistant to arsenite. One possibility is the toxicity of the ArsB protein in E. coli [9] since in the absence of IPTG, clone carrying only parsB grew as well as the will type (data not shown). On the other hand, parscbsB did not induce toxicity in E. coli and clone was arsenite resistant. It is possible that the co-transcription of CBS is required for a correct function of ArsB. The cbs gene encodes a protein with a CBS domain that probably dimerizes to form a stable globularstructure with ArsB [35].

Gene induction experiments

RT-PCR was used to test if each ars gene cluster forms a unique transcriptional unit. RT-PCR products related to all ars1 genes were obtainedshowing that genes were transcribed from one independentmRNA (Fig. 5). The RT-PCR experiments for ars2 also showed products for all genes demonstrating that all genes were transcribed from the same operon (data not shown).

Figure 5

RT-PCR analysis of ars1 genes of O. tritici SCII24. Total RNA isolated from O. tritici cells in the exponential phase was used as template in a reverse transcriptase reaction using the reverse primer from arsB to generate cDNA. Then, the several intergenic regions were amplified: arsR-arsD (ane1), arsD-arsA (lane2), arsD-cbs (lane3) and cbs-arsB (lane 4).

Detection of

Specific probes for structuralars genes (arsA, cbs domain, arsB, arsC1, Acr3, arsC2 and arsH) were designed to determine the presence of the ars genes in other strains belonging to the genus Ochrobactrum. Total DNA from these strains was analyzed by Southernblot (Fig. 6). The genes arsA, arsB and the gene coding for the CBS domain were only detected in strain O. tritici SCII24 and the arsC1 gene was detected in both strains of O. tritici (SCII24 and 5bvl1). Using the Acr3 probe a hybridizationsignal was obtained from preparations of O. tritici (SCII24 and 5bvl1) and type strain O. anthropi. Southern blot experiments performed with arsC2 and arsH probes yielded a signal for all of the Ochrobactrum strains tested except for type strain O. intermedium.

Discussion

The ability of strain O. tritici to resist up to 50 mM As(III), 10 mM Sb(III) and more than 200 mM As(V), turns this bacteriuminto one of the most resistant microorganisms described to date, most probably due to the presence of two functionalars operons (ars1 and ars2) even if the presence of other mechanisms in the cell, participating in arsenic resistance, can not be overruled. The ars1 operon conferred resistance to arsenite and antimonite and ars2 was responsible for resistance to arsenite and arsenate. Therefore, O. tritici is the first bacteria characterized which contains 2 operons involved in arsenic resistance each one conferring resistance to different metals or metal states. Although, several arrangements of arsenical resistance operons are possible in Bacteria, usually the genes that confer resistance to As(III) and As(V) are present in the same operon [reviewed in [15,16,36]]. In O. tritici the ars operons showed an unusual genetic composition. The organization of ars1 operon displayed some similarities with the well-known ars operon on the plasmid pR773 [7] and pR46 of E. coli [8], including the genes arsR, arsD, arsA and arsB. However, the arsC gene (arsenate reductase) was not identified in the O. tritici ars1 operon. A second atypical feature was the presence of one additional ORF usually not associated with ars genes, which codes a protein with a CBS domain. CBS domains are small domains of unknown function, which were only reported on ars genes cluster of Acidithiobacilluscalduspresent on a Tn21-like transposon [26] and on transposon, TnLfArs of Leptospirillum ferriphilum [27].

The ars2 operon of O. tritici could be also differentiated from previously described ars operons. One of the differences was the presence of two arsR genes with oppositeorientations. While arsR2 gene was located upstream with the same orientation as arsC1Acr3C2H, the arsR3 was located downstream and was divergently transcribed. Another intriguingcharacteristic was the presence of two arsenate reductase genes, arsC1 and arsC2, encoding distinct ArsC protein families. The ArsC1 belong to the family represented by the E. coli pR773 ArsC that uses glutathione and glutaredoxin as electron sources [37]. In contrast, the ArsC2 belong to the family represented by the S. aureuspI258 ArsC that uses thioredoxin as an electron source [38]. The arsR is commonly found in ars operons and its regulatory function, towards the basal operon expression, has been demonstrated [5,9,30]. For most of these trans-acting ArsR regulatory proteins, even with proteins with low homology, the consensus sequence for metal binding (ELC32VC34DLC37) was found. However, the three ArsRs from O. tritici had a very low or even no homology between them and they did not show the typical arsenite-binding motif (Fig. 2). The absence of conserved metal binding box was also previously identified in Corynebacterium glutamicum [29] and in Acidithiobacillus ferroxidans [20]. In the case of A. ferroxidans, Qinet al. [39] proved the involvement of a vicinal cysteinepair, Cys95, Cys96 and a third residue Cys102, in the transcriptional regulation by trivalentmetalloids. In O. tritici, it was apparent that ArsR1 could function as a repressor since the expression of the arsA gene (visible from SDS polyacrylamide gel) depended on the presence of the inducer. The most probable regulatory role of the remainingarsRs genes could not be evaluated, because the ars2 operon products were not detected by polyacrylamide gel. The arsD, together with the arsR, has been associated to the control of the maximal level of ars operon expression preventing the overexpression of ArsB [40], which is toxic in excess. Ourresults also illustrate the toxic effect of ArsB, since the overproduction of this arsenite efflux pump (ArsB) had a deleterious effect on E. coli cells. The experimental data of this work do not support the regulatory function of the ArsD over ArsB expression, since E. coli cells that did not carry the arsD gene (constructs parsAcbsB and parscbsB) showed similar resistance as the constructs with arsD, although the role of metallochaperone-mediator can not be excluded. The arsB gene product could not be detected in E. coli by SDS-PAGE as was previously reported by other authors [9]. Nevertheless, in contrast to other operons [4,41], the O. tritici arsB gene alone was not able to confer resistance to arsenite and antimony. However, the presence of the CBS domain in addition to arsB gene was enough to increase the resistance to these metalloids. CBS domains are found in a widerange of other unrelated proteins and in some of them a regulatory role of the protein structure has been suggested [35]. Therefore, in O. tritici it is possible that the protein with identity to CBS-binding domain mayplay a role as a structure decreasing the toxicity of the ArsB. As we mentioned before the ars2 operon also conferred resistance to As(III), although the ars2 operon was the only one responsible for arsenate detoxification. A second arsenite efflux pump was located in the ars2 operon, but, differently from arsB, this one was phylogenetically near to arsenite transporter familly-ACR3, represented by Saccharomyces cerevisiae [18]. O. tritici ACR3 protein as well as yeast Acr3p catalyzes extrusion of arsenite from the cells conferring arsenite but not antimonite resistance. As expected, ACR3 was not homologous to the ArsB found in operon ars1, even though they both function as arsenite efflux pumps.

Arsenate resistance in O. tritici could involve only one or both arsC genes to convert As(V) to As(III), since we observed that not only the ArsC1 was functional in E. coli, but also the ArsC2 usually related to gram-positive ArsC family. ArsC reductases from different phylogeneticgroups were already showed to be functional in E. coli [41]. The arsenite produced was then pumped out from the cells by ACR3 protein. An arsH-like gene was also located in operon ars2. The gene arsH was first described as a regulator [19] but no specific role could be conferred to arsH in A. ferroxidans [20] and in plasmid R478 of S. marcescens [21]. Recently, in Sinorhizobium meliloti [24] and in Shigella flexneri [25], ArsH was described as a H2O2-forming NADPH:FMN oxidoreductase that also reduces azodyes. O. tritici arsH gene did not have any effect in arsenate resistance and was not fundamental to arsenite resistance, however, the removal of this gene resulted in a reduction of arsenite resistance by E. coli cells in the presence of high levels of As(III). Therefore, a similar role to that proposed for the Sinorhizobium ArsH can be expected since homology studies of ArsH from O. tritici showed that the protein belongs to the same NADPH-dependent FMN reductase family.

Southern blotting did not revealars1-homologous sequences in the other type strains of the genus Ochrobactrum, but signed the presence of the ars2 genes in some of these bacteria. It is interesting to note the concurrent absence of the first cluster of genes along with strain-sensitivity to As(III) and Sb(III), suggesting the involvement of ars1 operon in detoxification of both oxyanions in these strains. In contrast, the detection in Ochrobactrum strains of genes from ars2 operon along with arsenate resistance may reflect the relation between the presence of ars2 genes and arsenate resistance.

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Conclusion

This work illustrates the presence of operons ars1 and ars2 in O. tritici and its possible involvement in arsenic resistance showed by E. coli heterologouscomplementation analysis. Operon ars1 seems to code for the main arsenite detoxification system and is entirely responsible for antimonite resistance in this strain. Operon ars2 encodes a group of different proteins with the arsenate detoxification as main function, and additionally is involved in arsenite resistance. This operon includes genes coding for arsenate reductases with different phylogenetic origins.

Arsenic and antimony resistance assays

Arsenite and antimonite resistance assays were carried out in LB medium, while for arsenate resistance, cells were grown in low phosphate medium. Overnight cultures were diluted 100-fold into fresh medium containing the ampicillin and different concentrations of arsenite, antimonite and arsenate. The bacterial suspensions were incubated at 37°C with shaking for 5 hours for experiments in LB medium or for 15 hours for assays performed in low phosphate medium and the absorbance at 600 nm was measured.

Southern hybridization

Southern blot analysis was performed as described by Sambrook et al. [42]. Purified DNAs (10 μg) of strains O. tritici SCII24, O. tritici 5bvl1, O. grignonense OgA9a, O. anthropi LMG 3331 and O. intermedium LMG 3301 were subjected to digestion with restriction enzymes and electrophoresed on agarose gel [0.8% (w/v)]. DNA was capillarytransferred for approximately 16 h to a nylon membrane in 0.4N NaOH, 1 M NaCl buffer followed by neutralization. DNA probes for structural genes [arsA, cbs domain, arsB, arsC1, Acr3, arsC2 and arsH] were amplified by PCR using gene-specific primers, recovered from agarose gels and purified with Wizard SV Gel and PCR Clean-Up System (Promega). Each probe was labelled with dioxigenin-dUTP nonradioactive (Roche Molecular Biochemicals) and the subsequent pre-hybridization and hybridization with the membrane were performed at 45°C to 58°C using DIG High Prime DNA Labelling and Detection Starter Kit II, following the manufacturer's instructions. The membrane was washedtwice with 2× SSC-0.1% SDS at roomtemperature and twice with 0.2× SSC-0.1% SDS for 15 min at 68°C by constantagitation. Membranes were autoradiographed after incubation at 37°C for 45 min.

Nucleotide sequence accession numbers

The nucleotide sequences of the DNA fragments containing the ars1 operon and ars2 operon have been submitted to GenBankunder the accession no. DQ490089 and DQ490090, respectively.

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